Fibroblast growth factor (FGF) is a series of multi-functional polypeptides, and 18 mammalian fibroblast growth factors (FGF1-FGF10, FGF16-FGF23) have been found. According to the different sequence homology, they are sub-divided into 6 subfamilies (Andrew Beenken et al., Nat Rev Drug Discov, 2009, 8(3): 235-253). Fibroblast growth factor 21 (FGF-21) was found for the first time in 2000 by Nishimura et al., (Nishimura T. et al., Biochim Biophys Acta, 2000, 1492: 203-206) from mice embryo. It belongs to the same subfamily as FGF-19 and FGF-23, does not bind heparin, and plays a part in endocrine regulations, such as metabolism of bile acid, cholesterol, glucose and vitamin D, etc.
Human FGF-21 gene is located at chromosome No. 19. The full length of FGF-21 comprises mature FGF-21 protein of 181 amino acids and a signal peptide of 28 amino acids at the N-terminal. Both N- and C-terminal of mature human FGF-21 protein have biological functions: N-terminal amino acids participate in the interaction with FGF receptors; whereas C-terminal amino acids participate in the interaction with beta-Klotho protein (Micanovic R. et al., J Cell Phys, 2009, 219(2): 227-234). Studies on the tissue cell distribution of FGF-21 are mainly at mRNA level, wherein endogenic FGF-21 mRNA was found first in thymus and liver, and then in pancreas, grease as well as in muscular tissues (Inagaki T. et al., Cell Metab, 2007, 5 (6): 415-425). The biological function of human FGF-21 was found while screening novel proteins potentially useful in diabetes treatment by glucose uptake assay (Kharitonenkov A. et al., J Clin Invest, 2005, 115 (6): 1627-1635). Recombinant human FGF-21 protein may stimulate the glucose uptake of differentiated mouse 3T3-L1 cell and human preadipocyte. Being different from the rapid insulin-intermediated utilization of glucose, the glucose uptake effect of FGF-21 can last for a few hours. More importantly, the glucose uptake with FGF-21 is non-insulin dependent, and can be enhanced in the presence of insulin (Alexei Kharitonenkov et al., Biodrugs, 2008, 22 (1): 37-44). Experiments show later that FGF-21 increases the non-insulin dependent glucose uptake by increasing the expression of glucose vectors-1 (GLUT-1) in 3T3-L1 cell and human preadipocyte (Amer P. et al., FEBS Lett, 2008, 582 (12): 1725-1730). Wente W. et al., discovered that (Wente W. et al., Diabetes, 2006, 55: 2470-2478) in isolated mouse pancreas islet cell and INS-1E cell, FGF-21 could increase the transcription, expression and secretion of insulin, thereby preventing programmed cell death intermediated by glycolipid and cell factors, protecting the amount and function of pancreas islet β-cell, as well as inhibiting glucose-mediated release of glucagon. In transgenic mice with high expression of FGF-21 (wherein the concentration of FGF-21 mRNA is 50-150 folds of that of the control), contents of the serum cholesterol, glucose and triglyceride in vivo are decreased significantly, as well as the fasting insulin level; also improved are insulin sensitivity and glucose clearance (Inagaki T, Cell Metab, 2007, 5 (6): 415-425). In contrast, severe metabolism abnormality is found in mice with adenovirus-mediated FGF-21 gene silence, including fatty liver, hypertriglyceridemia, significant increase of free fatty acids and total cholesterol content in serum, as well as decrease of ketones in serum, etc. (Badman M K et al., Cell Metab, 2007, 5 (6): 426-437). In animal models of diabetes of non-human primates, the content of LDL cholesterol could be decreased and HDL cholesterol increased by injecting recombinant human FGF-21, thus reducing risks of suffering from cardiovascular diseases (Kharitonenkov A. et al., Endocrinology, 2007, 148: 774-781). No hypoglycemia, oedema or obesity increase or other side effects is found in animal pharmacodynamic experiments of recombinant human FGF-21 in vivo, demonstrating its promising application prospect and safety.
Owing to its potential value for the treatment of diabetes, relevant drugs of human FGF-21 are now being studied by Lilly and Ambrx Corp. A recombinant human FGF-21 mutant (LP10152) is at phase I clinical trial in Lilly Corp. (Frye C C et al., WO2006/028595), whereas a PEG-modified human FGF-21 protein is on preclinical study in Ambrx and Merck Corp. (Thomas P. et al., US2008/025045).
Biological effects are retained in human FGF-21 with 4 amino acids deleted from the N-terminal (Micanovic R. et al., J Cell Phys, 2009, 219(2): 227-234); also, no decrease of bioactivity is found after local amino acid mutation or (and) deglycosylation (Frye C C et al., WO2006/028595). Therefore, it is now an important part in human FGF-21 research to modify the amino acid sequence.
GLP-1
As a post-translation product of proglucagon gene, glucagon-like-peptide-1 (GLP-1) was found for the first time in 1984 (Majaov S. et al., J Biol Chem, 1986, 261: 11880-11889). The human proglucagon gene is located at chromosome No. 17. It can be expressed not only in pancreatic α-cell, but also in intestinal L-cell, wherein products of genetic transcription and translation are the same and those of post-translation different. In pancreatic α-cell, proglucagon is cleaved into glucagon, enteroglucagon-associated peptides and a major fragment of proglucagon. In intestinal L-cell, proglucagon is cleaved into glucagon-like-peptide-1 (GLP-1), glucagon-like-peptide-2 (GLP-2) and enteroglucagon (Nielsen L L et al., Regul Pept, 2004, 117: 77-88).
The initial GLP-1 produced in intestinal canal is an inactive 37-peptide, wherein a 6-peptide at the N-terminal need to be removed to form GLP-1 (7-37), which has biological activity; and the C-terminal Gly can serve as a substrate of amidase and the peptide is further degraded into GLP-1 (7-36) amide with increased stability in vivo. About 80% of naturally occurring GLP-1 in intestinal canal exists with the latter form (Kieffer T J et al., Endocr Rev, 1999, 25: 876-913). The intestinal L-cell is stimulated to secrete GLP-1 in intestinal canal during food digestion, among which the stimulation of sugar and fat work the strongest. Normally, the concentration of plasma GLP-1 rises significantly at 5-30 minutes after a meal. As an important incretin, GLP-1 mainly possesses with following biological functions, including: (1) binding the specific receptor GLP-1R at pancreas islet β-cell to stimulate the synthesis and release of insulin in correspondance with glucose concentration: the higher the latter, the stronger the former, and vice verse; (2) inhibiting the release of glucagon in pancreas islet α-cell, thus reducing that of glycogen in livers; (3) increasing insulin sensitivity to improve the proliferation and function of pancreas islet β-cell, and to reduce apoptosis; (4) promoting differentiation of precursor pancreas islet cell into mature pancreas islet cell; (5) reducing the rate of stomach evacuation to inhibit appetite (Vaidya, H B et al., Curr Drug Targets, 2008, 9: 911-920).
With many advantages in treating diabetes, however, natural GLP-1 has limited clinical usage because of its short plasma half-life in vivo. A di-peptide is cleaved from the N-terminal of GLP-1 by dipeptidyl peptidase IV (DPP IV), which can specifically recognize Ala2 at the N-terminal; thus degrading GLP-1 into inactive GLP-1 (9-36) and GLP-1(9-37), and making its half-life to be only about 2 minutes. To prolong the half-life of GLP-1 in vivo for better clinical application, studies are now focused on structural improvement of GLP-1 for mutants specifically resisting to the degradation of DPP IV (Arulmozhi D K et al., Eur J Pharm Sci, 2006, 28: 96-108).
Exenatide, which is developed from GLP-1 by Amylin and Lilly Corp., USA, is the first drug against diabetes derived from GLP-1 receptor. Exenatide is a synthetic Exendin-4, which is an effective GLP-1R agonist of a short peptide of 39 amino acids found in Heloderma horridurn venom (Eng J et al., J Biol Chem, 1992, 267: 7402-7405). As a GLP-1 analogue, Exendin-4 shares 53% homology with GLP-1 and has Gly2 at the N-terminal, resisting to the degradation from DPP IV and prolonging the plasma half-life. Gly22 of GLP-1 breaks the space structure of helix, while Glu16 of Exendin-4 stabilizes the α-helix. Furthermore, the C-terminal of Exendin-4 comprises 9 amino acids that do not exist in GLP-1, making it to be unlikely digested by Endonuclease, and to be allowed to bind to the N-terminal of GLP-1R, hence enhancing the affinity (Kolterman O G, J Clin Endocrinol Metab, 2003, 88: 3082-3089). Owing to its structural advantages, Exendin-4 performs 5530-fold better than GLP-1 in lowering the level of blood sugar. Additionally, ZP10, a GLP-1 analogue derived from Exendin-4 wherein the C-terminal of Exendin-4 is added with 5-lysine (Petersen J S, Diabetologia, 2002, 45: A147), also exhibits prolonged half-life in blood, and therefore is allowed to be administrated once a day.
Liraglutide, an amidated GLP-1 derivative developed by Novo Nordisk Corp. in Denmark (Vilsbøll T, Diabetes Care, 2007, 30: 1608-1610), has Lys34 of GLP-1 substituted with Arg, as well as a C16 fatty acid connected to the Lys26 via glutamyl. Liraglutide can bind non-covalently to albumin in plasma and retain the antagonism activity of GLP-1 against DPP IV degradation, and has a prolonged half-life of 11-15 hours that is suitable for administration once a day (Elbrond B, Diabetes Care, 2002, 25: 1398-1404). Another GLP-1 analogue, CJC-1131, can bind covalently to Cys34 of albumin in plasma after modification of Lys37 at the C-terminal in vitro, thus leading to administration once a day (Kim, J G, Diabetes, 2003, 52: 751-759). It follows that bioactivity can be well retained with substitution, deletion, and addition of (one or several) amino acids at the C-terminal of GLP-1 or exendin-4 by chemical or genetic engineering methods.
Many patent publications and literatures about GLP-1 and human FGF-21 (such as Christopher P. Prior, PCT/US03/26818; Craig A Rosen, US2006/0194735A1; Wolfgang Glaesner, US2007/0036806, PCT/US04/16611; Thomas P. CUJEC, US2008/0255045A1) show that, with fused expression of GLP-1 or FGF-21 together with human blood albumin, Fc fragment of human IgG or human transferrin, respectively, (GLP-1-HAS, GLP-1-Fc, GLP-1-Transferrin, FGF-21-HAS, FGF-21-Fc), the half-life of GLP-1 or FGF-21 in vivo can be prolonged to achieve injection once a week at most. Also, PEG-GLP-1 and PEG-FGF-21 which are chemically modified with PEG can achieve a better half-life in vivo.
In conclusion, GLP-1 and FGF-21 are polypeptide drugs against diabetes that are nowadays paid much attention; the former has clear structure and function as well as pharmaceuticals granted by FDA; and the latter was found for the first time in 2004 to be antidiabetic and hypolipemic in animal experiments. Clinically, hyperglycemia occurs often with hyperlipidemia, however, there are no drugs that can simultaneously regulate plasma glucose and lipid, and possess with longer half-lives.